Sindbis virus is a species of the Alphavirus genus which includes such important pathogens as Venezuelan, Western and Eastern equine encephalitis viruses. In nature, Sindbis virus is transmitted by mosquitos and its alternate vertebrate host is usually a bird or a mammal. In vitro, Sindbis virus infects a variety of avian, mammalian, reptilian, and amphibian cells. It also infects many species of mosquitos, a tick, and Drosophila melanogaster. Infection of vertebrate cells in culture is usually characterized by a dramatic cytopathic effect and rapid cell death, whereas growth in mosquito cells often leads to the establishment of chronic or presistent infections.
Since its isolation in 1952, Sindbis virus and the closely related Semliki Forest virus have been widely studied (Schlesinger, S., and Schlesinger, M. J., 1986). The genome of Sindbis virus consists of a single molecule of single-stranded RNA, 11,703 nucleotides (nt) in length (Strauss, E. G. et al., 1984; Strauss, E. G. and Strauss, J. H., 1986). The life cycle of Sindbis virus is shown in FIG. 2. The genomic RNA is infectious, is capped at the 5' terminus and polyadenylated at the 3' terminus, and serves as mRNA and is therefore, by convention, of plus (+) polarity. The 5' two-thirds of the genomic 49S RNA is translated during early infection to produce two polyproteins that are processed by co-translational or post-translational cleavage into four nonstructural proteins (called nsPl through nsP4, numbered in order as they appear in the genome sequence; Strauss, E. G. et al., 1984) presumably required for RNA replication. A full-length minus strand complementary to the genomic RNA is then synthesized. This minus strand then serves as a template for the synthesis of new 49S genomic RNA molecules and as a template for transcription of a 26S subgenomic mRNA molecule. Transcription of the minus strand begins at an internal site to produce the 26S subgenomic mRNA that is 4,106 nt long and colinear with the 3' terminal one-third of the 49S genome. The subgenomic mRNA is capped and polyadenylated. It does not serve as a template for minus strand synthesis, nor is it packaged into mature virions. Translation of the 26S mRNA produces a polyprotein that is cleaved co- and post-translationally by a combination of viral and presumably host-encoded proteases to give the three virus structural proteins, a capsid protein (C) and the two envelope glycoproteins (El and PE2, the precursors of the virion E2). The capsid protein complexes with the genomic RNA to form intracellular icosahedral nucleocapsids. These nucleocapsids interact with the cytoplasmic domains of the transmembrane envelope proteins, resulting in the budding of virus at the plasma membrane.
Three features of Sindbis virus suggest that it would be a useful vector for the expression of heterologous (e.g. foreign) coding sequences. First, is the previously discussed wide host range of Sindbis virus, both in nature and in the laboratory. Second, Sindbis virus gene expression occurs in the cytoplasm of the host cell and is rapid and efficient. During the 8-12 hours of a typical infection at 37.degree. C., some 10.sup.7 to 10.sup.8 molecules of viral structural proteins are synthesized by each infected cell. Third, temperature-sensitive mutations in RNA synthesis are available that may be used to modulate the expression of heterologous coding sequences by simply shifting cultures to the non-permissive temperature at various time after infection.
Although the complete genomic sequence of Sindbis virus is known (Strauss, E. G. et al., 1984) and the coding regions for the viral proteins have been identified, the cis-acting sequences required for replication and transcription of infectious RNA molecules have heretofor not been identified. Utilization of Sindbis virus as an expression vector requires a determination of those virus sequences which allow for replication, transcription, and, ideally, packaging of the virus RNA before a heterologous coding sequence can be inserted into the virus RNA molecule.
Studies aimed at identifying conserved sequences among the alphavirus group have led to speculations as to the possible role of such conserved sequences in virus replication and transcription (see for review Strauss, E. G. and Strauss, J. H., 1986, and Ou, J-H. et al., 1982). No studies have yet been conducted, however, demonstrating the functionality of these conserved sequences in infectious RNA molecules. Furthermore, no suggestion has been made as to how or even if these conserved sequences can be employed to construct recombinant Sindbis virus vectors useful in expressing heterologous coding sequences. It remains to be determined how such virus sequences can be operably joined to heterologous coding sequences to provide for the replication, expression and/or packaging of heterologous coding sequences in infected host cells.
It is therefore an object of the present invention to identify the site(s) within the Sindbis virus genome where a heterologous coding sequence can be inserted without disrupting the ability of the recombinant molecules thereby created to be replicated and transcribed and, ideally, packaged by host cells containing such a recombinant molecule(s).
It is a further object of the present invention to construct Sindbis virus molecules (e.g., vectors) that contain at least one heterologous coding sequence, which molecules can provide for the replication and transcription (e.g., expression) of the heterologous coding sequence(s) in infected cells.